luteinizing hormone-releasing hormone (sGnRH-A)

Comparison of [ D - A ~~ ~r ~~,~'e ,u ' ,pro9NEt]-luteinizing hormone-releasing hormone (sGnRH-A), and [ D - A I ~pro9 ~ , NEt]-luteinizing hormone-releasing hormone (LHRH-A), in combination with pimozide, in stimulating gonadotropin release and ovulation in the goldfish, Carassius auratus Department of Zoology, Universi5 of Alberta, Edmonton, Altcr . , Canada T6G 2E9 AND

Can. J. Zool. Downloaded from www.nrcresearchpress.com by Renmin University of China on 05/28/13 For personal use only.

J. E. RIVIERA N D W. W . VALE Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, Sun Diego, C A , U.S.A. 92138 Received August 18, 1986

PETER, R. E., SOKOLOWSKA, M., NAHORNIAK, C. S., RIVIER, J . E . , and VAI-E.W . W . 1987. Comparison of [ D - A ~~ ~ r p~' , , ~ e u pro9 ~ , NEt]-luteinizing hormone-releasing hormone (sGnRH-A), and la', pro' NEt]-luteinizing hormonereleasing hormone (LHRH-A), in combination with pimozide, in stimulating gonadotropin release and ovulation in the goldfish, Carassius auratus. Can. J . Zool. 65: 987-991. LHRH-A and sGnRH-A were tested, in the presence or absence of pimozide (Pim), for their ability to stimulate increases in serum gonadotropin (GtH) levels and ovulation in prespawning female goldfish. In the absence of Pim, sGnRH-A was more active than LHRH-A in terms of stimulating serum GtH levels; neither peptide given alone was effective in stimulating ovulation. Pirn potentiated the activity of both peptides; high dosages of either peptide, plus a high dosage of Pim, were highly effective in stimulating serum GtH and ovulation. A low dosage of sGnRH-A, plus a low dosage of Pim, were also effective in stimulating serum GtH and ovulation; however, a low dosage of LHRH-A plus a low dosage of Pim were ineffective. The time to ovulation following injections, for those treatments that were highly effective in inducing ovulation, was highly predictable, and oocyte fertility and viability were high. These results indicate that sGnRH-A, in the presence of Pim, is effective in stimulating ovulation in goldfish at dosages about 10-fold less than for LHRH-A. The basis for the differences in potency between sGnRH-A and LHRH-A is discussed.

PETER, R. E . , SOKOLOWSKA, M., NAHORNIAK, C. S., RIVIER, J . E., et VALE,W. W. 1987. Comparison of [ D - ~ r g~~r ,p ' ,~ e u ' , pro9 NEt]-luteinizing hormone-releasing hormone (sGnRH-A). and [ D - ~ l a pro' ~ . NEt]-luteinizing hormone-releasing hormone (LHRH-A), in combination with pimozide, in stimulating gonadotropin release and ovulation in the goldfish, Carassius auratus. Can. J . Zool. 65 : 987-99 1 . Des tests en presence ou en l'absence de pimozide ont permis d'estimer la capacitk de I'hormone liberatrice de l'hormone luteinisante (LHRH-A) et de I'hormone liberatrice de la gonadotropine (sGnRH-A) de stimuler les augmentations de gonadotropine (GtH) serique et de dkclencher l'ovulation chez des femelles du poisson rouge juste avant la fraye. En l'absence de pimozide, l'hormone sGnRH-A stimule plus la gonadotropine serique que l'hormone LHRH-A; ni I'un ni l'autre de ces peptides ne peut dkclencher l'ovulation en l'absence de pimozide. Le pimozide augmente l'efficacite des deux peptides; des doses fortes de l'un ou l'autre peptide et une dose forte de pimozide stimulent fortement l'augmentation de GtH skrique et declenchent l'ovulation. Une faible dose de sGnRH-A et une faible dose de pimozide stimulent Cgalement la gonadotropine skrique et l'ovulation; cependant, une faible dose de LHRH-A et une faible dose de pimozide restent sans effet. Apres les traitements qui stimulent fortement l'ovulation, le temps de decalage avant l'ovulation est tres previsible et, de plus, la fertilite des ovocytes et leur viabilite sont elevees. Ces resultats indiquent que l'hormone sGnRH-A peut dkclencher efficacement l'ovulation en presence de pimozide 2des doses environ 10 fois moins klevees que dans le cas de la LHRH-A. Les facteurs de difference entre la puissance de I'hormone sGnRH-A et celle de la LHRH-A font l'objet d'une discussion. [Traduit par la revue]

Introduction In the goldfish, dopamine has been shown to act as a gonadotropin release-inhibitory factor (GRIF) by modulating the action of gonadotropin-releasing hormone (GnRH), as well by modulating the spontaneous release of gonadotropin (GtH) (Chang and Peter 1983a; Chang et al. 1984a, 1984b). Pimozide (Pim), a dopamine antagonist, potentiates the action of injected GnRH in goldfish (Chang and Peter 1983b; Chang et al. 1984b; Sokolowska et al. 1984, 1985a, 1985b; Peter et al. 1985) and in a number of other teleosts including several salmonids (for review see Peter et al. 1986). The practical importance of this is that treatment with the combination of Pim plus LHRH-A or another superactive LHRH agonist has been found to be highly effective ' ~ u t h o to r whom correspondence should be addressed. 'permanent address: Department of Ichthyobiotogy and Fisheries, Academy of Agriculture, ul. Ambrosowa 6, Krakow-Mydliniki, Poland.

for induction of ovulation in goldfish (Chang and Peter 1983a; Sokolowska et al. 1984, 1985a, 1985b), common carp (Billard et al. 1983; Lin et al. 1986a, 1986b), African catfish, Clarias lazera (de Leeuw et al. 1985a, 1985b), Chinese loach, Pararnisgurnus dabryanus (Lin et al. 1985, 1986c), silver carp, Hypophthalmichtys molitrix (Lin et al. 1986a, 1986b), bream, Parabramis pekinensis (Lin et al. 1986b), and mud carp, Cirrhinus molitorella (Lin et al. 1 9 8 6 ~ ) . The primary structure of a GnRH in chum salmon has been H Sherwood et al. determined as [ ~ r p ' , L ~ U ~ I - L H R(sCmRH; 1983). LHRH and sGnRH have similar potency in goldfish, in terms of magnitude and duration of the GtH release response, both in vitro (Mackenzie et al. 1984) and in vivo (Peter et al. 1985). Peter et al. (1985) found that superactivity of analogues of LHRH and sGnRH could be demonstrated in vivo in goldfish treated with Pim, and that sGnRH-A was the most potent analogue of all those tested; sGnRH-A was found to be 17 times more potent than LHRH-A in terms of the magnitude of serum

988

CAN. J. ZOOL. VOL. 65, 1987

GtH levels stimulated in male goldfish. The purpose of the present study was to compare the potency of LHRH-A and sGnRH-A in the stimulation of serum GtH levels and ovulation in prespawning female goldfish.

13 hours after injection l

A

o


Materials and methods Goldfish (Carassius auratus), common or comet varieties, were purchased from Ozark Fisheries, Stoutland, Missouri. Fish were held in 3200-L flow-through aquaria at 12- 15OC under simulated natural photoperiod and fed twice daily, to excess, with commercial trout pellets. Sexually mature (prespawning) females were selected for the experiment by gently squeezing the abdomen to eject a few oocytes from the ovipore, to confirm the presence of oocytes that had completed vitellogenesis , and acclimated to 12- 1 4OC and 16L:8D photoperiod in 150-L aquaria for 7- 14 days. Four days before an experiment females were individually tagged and weighed; at 10.- 17 h before the beginning of injections the water temperature was increased to 20°C. Pim, a gift from Janssen Pharmaceutica, Beerse, Belgium, was suspended in a vehicle of 0.7% NaCl and 0.1 % sodium metabisulphite, acidified with 0.2 mL glacial acetic acid per litre, and injected intraperitoneally (ip) at a dose of 1 or 10 pg/g body weight (g BWt). LHRH-A, purchased from Syndel Laboratories, Vancouver, B.C., Canada, and sGnRH-A, synthesized by J. Rivier and W . Vale as described by Habibi et al. (1987), were dissolved in acidified (pH = 6) fish physiological solution (PS; Burnstock 1958), and injected ip, either at the same time as Pim or 3 h later, at the doses of 0.1 or 0.0 1 pg/g B Wt. Injections were made in the evening, between 1700 and 2030. In the first and second experiments blood samples were taken at 13, 19, and 43 h after injection of the peptides by puncturing the caudal vasculature with a 25-gauge needle attached to a 1-mL syringe; in the third experiment blood samples were taken at 16 and 2 1 h after injections. Procedures for fish handling, blood sampling, and measurements of serum GtH levels by radioimmunoassay have been described (Peter et al. 1984). Ovulation was checked by stripping at the time of blood sampling, and additionally at 15, 16, 17, 22, 24, and 38 h after the second set of injections in experiment 1, and at 15, 16, 17, 18, and 24 h after injections in experiment 2. At the end of each experiment fish were killed by decapitation, and the occurrence of complete ovulation was confirmed by visual observation. To determine whether this technique for inducing ovulation has any influence on viability and development of embryos, ovulated oocytes from each ovulated fish in experiment 1 were stripped from the fish into a clean, dry petri dish within 1-2 h post-ovulation, and immediately fertilized with milt stripped from a spermiating male goldfish. After fertilization, several strands of nylon yarn, presoaked in aquarium water and identified according to donor fish, were laid in the petri dish and gently agitated to allow attachment of the fertilized eggs. The strands of yarn were then suspended in well-aerated aquarium water; a trace of malacite green was added to the water to retard fungal growth on the eggs. Viability of eggs was determined by randomly taking strands of yarn for each fish at about 26 and 50 h post-fertilization, and counting the number of living and dead embryos (about 50lstrand) under a dissecting microscope. The strands of yarn were discarded after examination. The Mann-Whitney U-test (two-tailed) was used to compare serum GtH concentrations between different treatment groups and the Fisher's exact test (one-tailed) was used to compare the number of goldfish that had ovulated between groups.

Results The first experiment (Fig. 1) tested the effects of pretreatment of prespawning female goldfish with Pirn (10 kg/g BWt) on the response to LHRH-A at a high dosage (0.1 kg/g BWt) and to sGnRH-A at low (0.01 kg/g BWt) and high (0.1 kg/g BWt) dosages. The control treatments of Pim, LHRH-A, and sGnRH-A (at both dosages) caused a significant increase in serum GtH levels compared with the Veh + PS control group at all sampling times; except for a significant difference in the serum GtH levels

300

A

vs Vehicle + PS vs Pimozide (10.0)-t PS vs Vehicle + sGnRH-A (0.01) vs Vehicle + sGnRH-A (0.1) vs Vehicle i LHRH-A (0.1) vs Pimozide (10.0)i LHRH-A T

19 hours after injection


60

1st Injection (-3hours)

T

Veh

1

Pirn Veh (10.0)

Veh 1

Veh

1

Pim Pim Pim (10.0)(10.0)(10.0) (0.01)

(0 hours) Ovulations

I 0/8

l 0/8

l 2/8

lj8

2118

I

4/8

l

718 + A

l

8/8 **

0 A

FIG. 1. Effects of injection of pimozide (Pim) and, 3 h later, LHRHA and sGnRH-A on serum gonadotropin levels (mean + SE) and ovulation in goldfish. Dosages (pg/g BWt) are given in parentheses. Statistical significance at p < 0.05 is indicated.

of the Pim (10 kg/g BWT) and LHRH-A (0.1 kg/g BWT) groups at 13 h, there were no significant differences in the serum GtH levels, or in the rates of ovulation between these four groups. Pirn in combination with LHRH-A (0.1 kg/g BWt) caused a significant increase in serum GtH levels compared with the Veh + PS control group at all sampling times; however, there were no significant differences in the serum GtH levels or ovulation rates between this group and the Veh plus LHRH-A group. Treatment with Pirn plus sGnRH-A at 0.0 1 or 0.1 kg/g BWt caused significantly higher serum GtH levels than in all other groups at the 13-h and 19-h sampling times; at the 43-h sampling time only the group treated with Pim plus sGnRH-A 0.1 kg/g BWt had higher serum GtH levels than all other groups. The ovulation rates of the two groups treated with Pirn plus sGnRH-A were significantly higher than in all other groups. The median (and range) in time to ovulation following the second set of injections for the Pirn (10 kg/g BWt) plus

989

PETER ET AL. 13 hours after injection

+

600


vs Veh~cle PS vs P ~ m o z ~ d(1e 0) PS A vs P ~ m o z ~ d (10.0) e PS vs Veh~clet L H R H - A (0.1) o vs Veh~cle s G n R H - A (0.01) A v s Vehlcle t s G n R H - A (0.1)

+ +

+


3 00

,

+

vs Veh~cle PS vs Pimozide (1.0) PS A vs Pimozide (10.0) PS vs Vehicle LHRH-A (0.01) o vs Vehicle sGnRH-A (0.01) A vs Pimoz~de (10 ) LHRH-A (0.01) +

+ +

n

T

E

+ +

+

300 21 hours after injection 2001


Veh

Plm

Plm

Veh

Veh

Pirn

Plm

Plm

Pim

0/7

0/7

4/8

5/7

8/8

(0.01)

Ovulations

200 r 42 hours after injection

0/8

0/8

0/8

0/7

+*

+o

FIG. 3. Effects of injection of pimozide (Pim) and LHRH-A and sGnRH-A on serum GtH levels (mean SE) and ovulation in goldfish. Dosages (kg/g BWt) are given in parentheses. Statistical significance atp < 0.05 is indicated.

*

FIG. 2. Effects of injection of pimozide (Pim) and LHRH-A and sGnRH-A on serum gonadotropin levels (mean SE) and ovulation in goldfish. Dosages (pg/g BWt) are given in parentheses. Statistical significance at p < 0.05 is indicated.

*

LHRH-A (0.1 p.g/g B Wt) group was 13 h ( 13- 16 h), for the Pim (10 p.g/g BWt) plus sGnRH-A (0.01 p.g/g BWt) group 16 h (13- 16 h), and for the Pim (10 p.g/g B Wt) plus sGnRH-A (0.1 pg/g B Wt) group 13 h ( 13- 16 h). Ovulated oocytes from each ovulated fish in expt. 1 were artificially fertilized within 1-2 h post-ovulation, and cultured to determine viability. There were no evident differences in viability of fertilized oocytes between the various treatment groups; at approximately 26 and 50 h following fertilization, 72% and 7 1%, respectively, of embryos were alive and appeared to be undergoing normal development. The second experiment (Fig. 2) tested the effects of simultaneous injection of prespawning female goldfish with Pirn at two dosages (1 and 10 p.g/g BWt), and LHRH-A (0.1 pg/g BWt) and sGnRH-A at two dosages (0.0 1 and 0.1 p.g/g B Wt). As in the first experiment, the control treatments of Pim, LHRHA, and sGnRH-A alone each caused significant increases in serum GtH levels, although none of the treatments was effective in causing a high rate of ohlation; the low dosage of Pim caused only a small increase in serum GtH levels compared with the effects of the high dosage of Pim. Notably, in contrast to the first experiment, the combination of Pirn ( 10 p.g/g B Wt) plus LHRHA (0.1 p.g/g BWt) stimulated significantly higher serum GtH levels than LHRH-A (0.1 p.g/g BWt) alone. Also, the ovulation

rate of the Pirn ( 10 pg/g B Wt) plus LHRH-A (0.1 kg/g B Wt) group was similar to the high rates of ovulation found in the Pirn (10 kg/g BWt) plus sGnRH-A (0.0 1 and 0.1 kg/g BWt) groups; the serum GtH levels of these three groups were similar at the 13-h and 19-h sampling times. The low dosage of Pim (1 kg/g BWt) plus sGnRH-A at both 0.01 and 0.1 kg/g BWt caused significantly higher serum GtH levels, at some sample times, than treatment with sGnRH-A alone at the same respective dosages. Treatment with Pirn (1 p.g/g BWt) plus sGnRH-A (0.1 kg/g BWt) caused a rate of ovulation that was lower than, but not significantly different from, the rates in the groups treated with Pirn (10 pg/g BWt) plus sGnRH-A (0.01 and 0.1 p.g/g BWt); the rate of ovulation induced by treatment with Pirn (1 p.g/g BWt) plus sGnRH-A (0.01 p.g/g BWt) was also not significantly different from the zero rate of ovulation in the Veh plus PS group. The median (and range) in time to ovulation for the Pim ( 10 p.g/g B Wt) plus LHRH-A (0.1 p.g/g B Wt) group was 13 h ( 13- 18 h), for the Pirn ( 1 p.g/g B Wt) plus sGnRH-A (0.01 p.g/g BWt) group 16 h (13- 16 h), for the Pim (1 p.g/g B Wt) plus sGnRH-A (0.1 pg/g B Wt) group 13 h ( 13- 18 h), for the Pim ( 10 p.g/g B Wt) plus sGnRH-A (0.0 1 p.g/g B Wt) group 13 h (1 3- 16 h), and for the Pim ( 10 p.g/g B Wt) plus sGnRH-A (0.1 p.g/g BWt) group 13 h (13-16 h). In the third experiment (Fig. 3) the effects of simultaneous injection of prespawning female goldfish with Pirn at two dosages (1 and 10 pg/g B Wt) and both LHRH-A and sGnRH-A at a low dosage (0.0 1 pglg B Wt) were tested. The control treatment of Pirn at 10 p.g/g B Wt and sGnRH-A at 0.0 1 p.g/g B Wt caused a significant increase in serum GtH levels; however, the responses were smaller in magnitude than in the previous experiments and there were no significant increases in serum GtH levels in response to Pirn at 1 pg/g B Wt and LHRH-A at 0.0 1 p.g/g B Wt. The responses to Pirn ( 1 and 10 p.g/g B Wt) plus sGnRH-A (0.0 1

990

C A N . J . ZOOL. VOL. 65. 1987


pglg BWt), in terms of serum GtH levels stimulated and ovulation rates, were similar to those of exp. 2. Although the serum GtH levels stimulated by Pim (10 pglg BWt) plus LHRH-A (0.01 pglg BWt) were significantly less than for Pirn (10 pglg BWt) plus sGnRH-A (0.01 kglg BWt), the ovulation rates induced by both treatments were similar. On the other hand, there was no apparent effect of LHRH-A at 0.01 pglg BWt when given in combination with Pim at 1 kglg BWt, as there was no significant stimulation of ovulation, and there were no significant differences in the serum GtH levels for the group compared with the group treated with Pim at 1 pglg BWt alone.

Discussion The results confirm that a single injection of LHRH-A at a high dosage (0.1 pglg BWt) causes a significant increase in serum GtH levels in prespawning female goldfish (Chang and Peter 1983a, 1983b; Sokolowska et al. 1984, 1985a, 1985b). A single injection of sGnRH-A, at low and high dosages (0.01 and 0.1 pglg B Wt, respectively), was also effective in stimulating an increase in serum GtH levels. Notably, sGnRH-A stimulated higher levels of serum GtH than similar dosages of LHRH-A, particularly at sampling times nearing 24 h or later after injection. Injection of Pim at a high dosage (10 kglg BWt) caused a significant increase in serum GtH levels in prespawning female goldfish, but the response was variable between experiments and was ineffective in inducing ovulation, as in previous studies (Chang and Peter 1983b; Sokolowska et al. 1984, 1985a, 1985b). Pirn potentiated the GtH release response to LHRH-A and sGnRH-A; Pim and other dopamine antagonists have previously been shown to potentiate the actions of LHRH-A and other LHRH agonists in goldfish and a number of other teleosts (for review, Peter et al. 1986). sGnRH-A plus Pim stimulated higher levels of serum GtH than similar dosages of LHRH-A plus Pim, in particular at sampling times nearing 24 h or later. This confirms earlier findings using male goldfish (Peter et ul. 1985) demonstrating that, in the presence of Pim, sGnRH-A is a more potent agonist than LHRH-A. A single injection of LHRH-A alone or sGnRH-A alone was ineffective in inducing ovulation in the goldfish, confirming earlier studies using LHRH-A (Chang and Peter, 1983b; Sokolowska et al. 1984, 1985a, 1985b). The combinations of Pim at a high dosage (10 kglg BWt) and LHRH-A and sGnRH-A at high dosages (0.1 kglg BWt) were about equally effective in inducing ovulation in the goldfish. A low dosage of Pirn ( I pglg BWt) plus low and high dosages of sGnRH-A (0.01 and 0.1 pglg BWt, respectively) were also effective in inducing ovulation, although the combination of Pirn (1 pglg BWT) and sGnRH-A (0.01 kglg BWT) was marginal; however, a low dosage of LHRH-A was only effective in combination with a high dosage of Pim. Overall, these results indicate that in terms of stimulation of GtH secretion and induction of ovulation, sGnRH-A is more potent than LHRH-A when combined with Pim. Also, for stimulation of serum GtH levels and ovulation, there appears to be no particular advantage in injection of Pirn 3 h prior to LHRH-A or sGnRH-A. In common carp, injection of Pirn at 0 , 3, or 6 h prior to LHRH-A resulted in a high rate of ovulation, whereas injections with a 10-h interval were ineffective (Lin et al. 1 9 8 6 ~ ) . The length of time from injection of Pim plus LHRH-A or sGnRH-A to ovulation in goldiish is reasonably predictable under the environmental conditions of the present experiments. Notably, the ovulated oocytes from the fish treated with Pirn

plus LHRH-A or Pim plus sGnRH-A were highly fertile and had high viability. The present results confirm studies by Lin et al. 1986a, 1986b) on silver carp and mud carp indicating predictability of the response time to ovulation, and high fertility and viability of oocytes from fish ovulated by treatment with Pirn plus LHRH-A. Substitution of certain D-amino acids in position 6 has been demonstrated to increase resistance to enzymatic degradation of LHRH analogues, as well as to add conformational stability and increase hydrophobicity, resulting in an increase in receptor affinity, all of which contribute to superactivity of the analogues (Nestor et al. 1984; Karten and Rivier 1986). sGnRH is hydrophobic compared to LHRH (Sherwood et al. 1983), and position 6 substitutions in sGnRH that further increased hydrophobicity did not further increase biological activity in goldfish (Peter et al. 1985). Studies on the in vivo activity in goldfish of various native vertebrate GnRHs and position 8 s~bstitutedanalogues of LHRH indicated that GnRH receptors in this species have very broad specificity (Peter et al. 1987). On this basis it is not possible at this time to ascribe the differences in potency between sGnRH-A and LHRH-A goldfish to specific structural features of either analogue. Habibi et al. ( 1987) found that the equilibrium association constant of sGnRH-A for high affinity binding sites in the goldfish pituitary was about 4.5-fold higher than for sGnRH. Perhaps sGnRH-A has a higher affinity than LHRH-A for pituitary GnRH receptors. The tendency, shown in the present work, for sGnRH-A to have a longer lasting action than LHRH-A suggests that a difference in metabolic inactivation may also exist. Studies are currently underway to evaluate these possibilities.

Acknowledgements This work was supported by grants to R. E. Peter from the Natural Sciences and Engineering Research Council of Canada and the International Development Research Centre of Canada. We wish to thank L. Wang for his assistance in performing these experiments. BILLARD, R . , ALAGARSWAMI, K . , PETER,R. E., and BRETON,B. 1983. Potentialisation par le pimozide des effets du LHRH-A sur la secretion gonadotrope hypophysaire, l'ovulation et la spermiation chez la Carpe commune (Cjiprinus c-arpio). C .R . Hebd. Seances Acad. Sci. Ser. 3 , 296: 18 1 - 184. BURNSTOCK, G. 1958. Saline for freshwater fish. J . Physiol. (London). 141: 35-44. CHANG.J . P., and PETER,R. E. 19830. Effects of dopamine on gonadotropin release in female goldfish, Curas.sius aurutus. Neuroendocrinology, 36: 35 1-357. ~~] 19836. Effects of pimozide and des Gly"', I D - A ~ luteinizing hormone-releasing hormone ethylamide on serum gonadotropin concentrations, germinal vesicle migration and ovulation in female goldlish, Carcrssins clurcztus. Gen. Comp. Endocrinol. 52: 30-37. CHANC;. J . P., MACKENZIE, D. S . . COULD,D. R . , and PETER.R. E. 1984n. Effects of dopamine and norepinephrine on in 19itro spontaneous and gonadotropin-releasing hormone-induced gonadotropin release by dispersed cells or fragments of the goldfish pituitary. Life Sci. 35: 2027-2033. CHANG, J . P., PETER,R. E.. N A H O R N I ACK. ,S . , and SOKOI~OWSKA, M. 1984b. Effects of catecholaminergic agonists and antagonists on serum gonadotropin concentrations and ovulation in goldlish: evidence for specificity of dopamine inhibition of gonadotropin sccretion. Gen. Comp. Endocrinol. 55: 351-360. DE LEEUW,K., GOOS,H . J . T H . .RICHTER, C . J . J . , and EDING, E. H . 1 9 8 5 ~Pimozide-LHRHa-induced . breeding of the African catfish. Clarias guriepinus (Burchell). Aquaculture, 44: 295-302.


PETER ET AL.

DE LEEUW,R., RESINK,J. W., ROOYAKKERS, E. J . M., and Goos, J. H. TH. 19856. Pimo~idemoddates the luteinizing hormonereleasing hormone effect on gonadotropin release in the African catfish, Clarias lazera. Gen. Comp. Endocrinol. 58: 120- 127. HABIBI, H. R., PETER,R. E., SOKOLOWSKA, M., RIVIER,J . E., and VALE,W. W. 1987. Characterization of gonadotropin-releasing hormone (GnRH) binding to pituitary receptors in goldfish (Carassius auratus). Biol. Reprod. In press. KARTEN,M. J., and RIVIER,J. E. 1986. Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: Rationale and perspective. Endocr. Rev. 7: 44-66. G., LIN,H. R., PENG,C., Lu, L. Z., ZHOU,X. J., VANDERKRAAK, and PETER,R. E. 1985. Induction of ovulation in the loach (Paramisgurnus dabryanus) using pimozide and [D- la^, pro9-Nethylamidel-LHRH. Aquaculture, 46: 333-340. LIN,H. R., LIANG,J. Y., PENG,C . , LI, G. Y., LIU,L. Z., ZHOU,X. J., CHANG, M. L., VANDERKRAAK, G., and PETER,R. E. 1986a. Pimozide and reserpine potentiate the effects of LHRH-A on gonadotropin secretion and ovulation in cultivated fishes in China. In Proceedings of the Asian Symposium on Freshwater Fish Culture. Edited by Board of Editors, J. Fisheries. Academic Publications, Beijing, China. In press. LIN,H. R., LIANG,J. Y., PENG,C . , LI, G. Y., Lu, L. Z., VANDER KRAAK, G., and PETER,R . E. 19866. The effects of LHRH analogue and drugs which block the effects of dopamine on gonadotropin secretion and ovulation in fish cultured in China. In Proceedings of the International Symposium on the Aquaculture of Carp and Related Species. Edited by R. Billard. INRA publications, Versailles, France. In press. LIN, H. R., PENG,C., VAN DER KRAAK,G . , PETER,R. E., and BRETON,B. 1 9 8 6 ~ .Effects of [ D - ~ l a ~Pro9-NE~]-LHRH , and catecholaminergic drugs on gonadotropin secretion and ovulation in the Chinese loach (Paramisgurnus dabryanus). Gen. Comp. Endocrinol. 64: 389-395. D. S., GOULD,D. R., PETER,R. E., RIVIER, J. E., and MACKENZIE, VALE,W. 1984. Response of superfused goldfish pituitary fragments to mammalian and salmon gonadotropin-releasing hormones. Life Sci. 35: 20 18-2026.

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